Papilloma virus vaccine

ABSTRACT

A method of providing papilloma virus like particles which may be used for diagnostic purposes or for incorporation in a vaccine for use in relation to infections caused by papilloma virus. The method includes an initial step of constructing one or more recombinant DNA molecules which each encode papilloma virus L1 protein or a combination of papilloma virus L1 protein and papilloma virus L2 protein followed by a further step of transfecting a suitable host cell with one or more of the recombinant DNA molecules so that virus like particles (VLPs) are produced within the cell after expression of the L1 or combination of L1 and L2 proteins. The VLPs are also claimed per se as well as vaccines incorporating the VLPs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.08/185,928, filed Jan. 19, 1994, incorporated herein by reference in itsentirety, which is a U.S. National Stage of International ApplicationPCT/AU1992/000364, filed Jul. 20, 1992, incorporated herein by referencein its entirety, which claims benefit of priority to AustralianApplication PK7322, filed Jul. 19, 1991, incorporated herein byreference in its entirety.

FIELD OF INVENTION

THIS INVENTION relates to papilloma viruses and in particular antigensand vaccines that may be effective in treatment of infections caused bysuch viruses.

BACKGROUND OF THE INVENTION

Papilloma virus infections are known not only in humans but also inanimals such as sheep, dogs, cattle, coyotes, wolves, possums, deer,antelope, beaver, turtles, bears, lizards, monkeys, chimpanzees,giraffes, impala, elephants, whales, cats, pigs, gerbils, elks, yaks,dolphins, parrots, goats, rhinoceros, camels, lemmings, chamois, skunks,Tasmanian devils, badgers, lemurs, caribou, armadillo, newts and snakes(see for example “Papilloma Virus Infections in Animals” by J P Sundbergwhich is described in Papilloma viruses and Human Disease, edited by KSyrjanen, L Gissman and L G Ross, Springer Verlag 1987).

It is also known (eg. In Papilloma viruses and Human Cancer edited by HPfister and published by CRC Press Inc 1990) that papilloma viruses areincluded in several distinct groups such as human papilloma viruses(HPV) which are differentiated into types 1-56 depending upon DNAsequence homology. A clinicopathological grouping of HPV and themalignant potential of the lesions with which they are most frequentlyassociated may be separated as follows.

In a first group may be listed types 1 and 4 which cause benign plantarwarts, types 2, 26, 28 and 29 which cause benign common warts, Types 3,10 and 27 which cause benign flat warts and Type 7 which causesbutcher's warts. This first group of infections occur in normal orimmunocompetent individuals.

In a second group which refer to immunocompromised individuals there maybe listed Types 5 and 8 which cause highly malignant macular lesions,Types 9, 12, 14, 15, 17, 19-25, 36 and 46-50 which cause macular or flatlesions which are benign or rarely malignant. These macular lesions areotherwise known as epidermodyplasia verruci formis (EV).

In a third group which infect particularly the genital tract there maybe listed Types 6, 11, 34 and 39, 41-44, and 51-55 which causecondylomata which are rarely malignant, Types 13 and 32 which causebenign focal epithelial hyperplasia, Types 16 and 18 which causeepithelial dysplasia and other lesions considerable potential includingbowenoid papulosis, and Types 30, 31, 33, 35, 45 and 56 which causecondylomata with intermediate malignant potential. The condylomataappear mostly in the anogenital tract and in particular the cervix.Types 16 and 18 are associated with the majority of in situ and invasivecarcinomas of the cervix, vagina, vulva and anal canal. The condylomatamay also occur in the aerodigestive tract.

In particular HPV16 is associated with premalignant and malignantdiseases of the genito-urinary tract, and in particular with carcinomaof the cervix (Durst et al., PNAS 80 3812-3815, 1983; Gissmann et al.,J. Invest. Dermatol 83 265-285, 1984). Presently, there is noinformation on the role of humoral responses in the neutralization ofHPV16.

The detection of antibodies against HPV16 fusion proteins (Jenison etal., J Virol 65 1208-1218, 1990; Rachel et al, Int. J. Cancer 48682-688, 1991) and synthetic HPV16L1 peptides (Dillner et al. Int. J.Cancer 45 529-535, 1990) in the serum of patients with HPV16 infectionconfirms that there are B epitopes within the capsid proteins of HPV,though few patients have HPV16 L1-specific antibodies identified bythese techniques. There is no system for HPV16 propagation in vitro, andhuman genital lesions produce few HPV16 virions; therefore HPV16particles have not been available for immunological studies.

The animal papilloma viruses may also include bovine papilloma virus(BPV) and in particular types BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6which are also differentiated by DNA sequence homology. In general theother animal papilloma viruses infect deer, horses, rabbits, dogs,rodents and birds. Papilloma viruses are small DNA viruses encoding forup to eight early and two late genes. (for review see Lancaster andJenson 1987 Cancer Metast. Rev. p 6653-6664; and Pfister 1987 Adv.Cancer Res 48, 113-147). The organisation of the late genes is simplerthan the early genes. The late genes L1 and L2 slightly overlap eachother in most cases. The putative L2 proteins are highly conserved amongdifferent papilloma viruses particularly the sequence of 10 basic aminoacids at the C-terminal end. The broad domain in the middle reveals onlysmall clustered similarities. The L10RF however appears monotonouslyconserved in all known cases. (See Syrjanen et al above). The amino acidsequence homology reaches 50% with the comparison between HPV1a, HPV6b,BPV1 and CRPV (Cotton tail rabbit papilloma virus).

In regard to immunotherapy concerning papilloma virus infections priormethods of treatment of warts and epithelial skin lesions have involvedthe use of surgery which can be painful and traumatic with scarringoften a result with the risk that reinfection can occur. Treatment withchemicals has also been used. A common treatment agent is salicylic acidwhich is the main ingredient in strengths ranging from 10% to 40% intinctures and plasters. Formalin in strengths of 38-20% has also beenproposed. Cryotherapy has been used for treatment of skin warts.Gluteraldehyde as a treatment agent has also been used. Podophyllin hasalso been used with varying success for both skin warts and anogenitalcondylomata. The types of surgery that has been used on anogenitalcondylomata has included surgical excision, cryosurgery and lasersurgery. The use of interferons has also been proposed (see Syrjanen etal above).

Antibodies to the L1 protein of bovine papillomavirus (BPV) havevirus-neutralization activity (Pilacinski et al., 1986) and HPV11virions can be inactivated in an in vitro model by specific antisera(Christensen and Kreider, J. Virol 64 3151-3156, 1990). There is alsosome evidence that spontaneous regression of HPV1-induced cutaneouswarts is associated with increased humoral immune responses to wartprotein (Kirchner, Prog. Med. Virol 33 141, 1986).

Vaccines have also been proposed with indifferent success. It has beenproposed to use vaccines containing autogenous tumor homogenates[Abcarian et al J. Surg Res 22: 231-236 (1977) Dis Colon Rectum25:648-51 (1982) Dis Colon Rectum 19: 237-244 (1976)]. However it hasrecently been advocated that patients should no longer be treated withautogenous vaccines because of the potential oncogenic effect of theviral DNA (Bunney 1986 Br Med J 293 1045-1047).

In relation to production of genetically engineered vaccines this matterhas been discussed in Pfister (1990) above and it seems that difficultyhas been experienced in obtaining an effective vaccine because of theplethora of different papilloma virus types. Pfister however points outthat attention should be directed to the so called early proteins (ie.E1, E2, E3, E4, E5, E6, E7 or E8) because these proteins are most likelysynthesised in the proliferating basal cells of a wart infection incontrast to the structural proteins which are expressed in the upperepidermal layers. Therefore according to Pfister (1990) virus capsidprotein appears to be limited in relation to use in a vaccine. The useof recombinant vaccinia viruses in in vitro test systems for papillomavirus early proteins in eukaryotic cells has been discussed also inPfister (1990). This may take the form of a live vaccine consisting ofgenetically modified vaccina virus expressing papilloma virus proteinsor on the surface of paraformaldehyde fixed autologous cells infected invitro with vaccinia recombinants or transfected with other expressionvectors. Another strategy for vaccine development as discussed inPfister (1990) is to use an immune stimulating complex of the glycosideQuil A.

Data on successful proplylactic vaccination exist only for bovinefibropapillomas homogenised homogenate of bovine fibropapillomas and hasbeen shown to provide limited immunity (Olson et al J Am Vet Med Assoc135, 499 (1959) Cancer Res 22 463 (1962)). A vaccine including anengineered L1 fusion protein (Pilacinski et al. UCLA Symp. Molecular andCellular Biology New Series Vol 32 papilloma Viruses Molecular andClinical Aspects Alan R Liss New York 1985 257) has also been used incalves but proved unsuccessful in humans (Barthold et al J. Am. Vet Med.Assoc. 165, 276, 1974). In Pfister (1990) it is stated that there ispresently no evidence for a possible prevention of HPV infection by theuse of a capsid protein vaccine, but induction of an antitumor cellimmunity appears to be feasible.

The L1 and L2 genes have been the basis of vaccines for the preventionand treatment of papilloma virus infections and immunogens used in thediagnosis and detection of papilloma viruses (International PatentSpecifications WO8605816 and WO830623). However, it appears that nocommercial usage of these vaccines have taken place.

SUMMARY OF THE INVENTION

Therefore it is an object of the invention to provide virus likeparticles (VLPs) which may be useful as diagnostic agents as well asforming a component of a vaccine for use with papilloma virusinfections.

The invention therefore in one aspect includes a method for productionof papilloma virus like particles (VLPs) including the steps of:

(i) constructing one or more recombinant DNA molecules which each encodepapilloma virus L1 protein or a combination of papilloma virus L1protein and papilloma virus L2 protein; and

(ii) transfecting a suitable host cell with said one or more recombinantDNA molecules so that virus like particles (VLPs) are produced withinthe cell after expression of the L1 or combination of L1 and L2proteins.

The invention in another aspect includes a vaccine containing thepapilloma virus VLPs in combination with a suitable adjuvant.

In relation to step (i) only papilloma virus L1 protein is required toform VLPs of some papilloma viruses. Suitably only the L1 protein isrequired to form VLPs of BVP1, HPV11 and HPV6 including HPV6b. HoweverVLPs may also be formed in relation to BPV1, HPV11 or HPV6b containingboth L1 and L2 proteins. For the formation of VLPs of other papillomaviruses such as HPV16, both the L1 and L2 proteins are required. Thissituation is also believed applicable to HPV18 which has similarpathological symptoms to HPV16 and also similar DNA sequence homology.Further it will be appreciated that the L1 and L2 genes may be includedin the same DNA recombinant molecule or in different DNA recombinantmolecules.

Preferably the recombinant DNA molecules are contained in recombinantvirus which may transfect the host cell. Suitable viruses that may beused for this purpose include baculovirus, vaccinia, sindbis virus,SV40, Sendai virus, adenovirus, retrovirus or poxviruses. Suitable hostcells may include host cells that are compatible with the above virusesand these include insect cells such as Spodoptera frugiperda, CHO cells,chicken embryo fibroblasts, BHK cells, human SW13 cells, drosophila,mosquito cells derived from Aedes albopictus or monkey epithelial cells.It will also be appreciated that other eukaryote cells may compriseyeast cells or other mammalian cells.

The DNA recombinant molecule is suitable obtained from a source ofpapilloma virus genome whereby L1 protein or L2 protein may be amplifiedby PCR amplification using suitably designed primers discussedhereinafter. Preferably a gene encoding L1 protein is inserted in aplasmid containing a suitable promoter and a DNA fragment containing theL1 protein and promoter is incorporated in a primary plasmid which mayconstitute the recombinant DNA molecule which may be inserted into arecombinant virus vector as described above.

A gene encoding the L2 protein may also be linked to a suitable promoterand preferably a DNA fragment incorporating the L2 gene and promoter isinserted into the primary plasmid to provide a doubly recombinantplasmid or secondary plasmid which plasmid may also be inserted in arecombinant virus vector as described above to form a doubly recombinantvirus vector.

However the invention also includes the embodiment wherein the primaryplasmid and/or the secondary plasmid may infect a suitable host cell toproduce VLPs containing L1 protein or VLPs containing L1 and L2 proteinunder appropriate experimental conditions. The latter VLPs are the idealimmunogen for a papilloma virus specific vaccine, as the L2 protein isimmunodominant in natural infection.

Other suitable DNA recombinant molecules include cosmids as well asrecombinant viruses. Suitable expression systems include prokaryoticexpression systems including E coli and any plasmid or cosmid expressionvector or eukaryotic systems including host cells described above incombination with a recombinant virus vector or alternatively yeast cellsand yeast plasmids.

In the situation where plasmids are used which incorporate genesencoding L1 or both L1 and L2 and wherein such plasmids may infect asuitable host cell for production of VLPs such plasmids should alsoinclude a suitable promoter to enhance expression of the VLP structuralproteins and a polymerase may also be utilised which is associated withthe relevant promoter. However in this situation VLPs may only beobtained under specific experimental conditions.

The L1 and L2 genes may be driven off any mammalian or viral promoterwith a mammalian or viral polyadenylation signal. Preferably the L1 andL2 genes are transcribed from any vaccinia virus promoter which may bean early promoter or late promoter as considered appropriate. A list ofsuch promoters is given in Davision and Moss (1989) J. Mol. Biol. 210749-769 and (1989) J. Mol. Biol. 210 771-784.

In the experimental work that has taken place the L1 gene is locateddownstream of a vaccinia 4b promoter and the L2 gene is locateddownstream of a synthetic vaccinia 28 k late promoter. The host cell ismonkey epithelial cells.

The VLPs may be obtained from the transfected cells by any suitablemeans of purification. The VLPs may be combined with any suitableadjuvant such as ISCOMS, alum, Freunds Incomplete or Complete Adjuvant,Quil A and other saponins or any other adjuvant as described for examplein Vanselow (1987) S. Vet. Bull. 57 881-896.

Reference may now be made to various preferred embodiments of theinvention as illustrated in the attached drawings. In these preferredembodiments it should be noted that the specific papilloma viruses, VLPsand specific constructs of DNA recombinant molecules are given by way ofexample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of plasmids used to construct HPV16recombinant vaccinia viruses.

FIGS. 2A and 2B are a western blot analysis of recombinant HPV16 L1 invaccinia virus infected CV-1 cells.

FIG. 3 is a northern blot analysis of recombinant vaccinia virusinfected CV-1 cells.

FIGS. 4A and 4B are electron microscopy of HPV virus-like particles fromCV-1 cells infected with recombinant vaccinia virus.

FIG. 5 illustrates CsCl equilibrium density gradient sedimentation ofHPV16 empty capsids.

FIG. 6 illustrates Glycosylation of L1 proteins in purified virusparticles.

FIG. 7 is a flow diagram of the constructions of plasmid pLC200 encodingL1 and pLC201 encoding L1 and L2.

FIG. 8 is a western blot analysis of the reactivity of murine sera withbaculovirus recombinant L1 protein.

FIGS. 9 and 10 illustrate mapping results for sera from BLAB/c, C57B1/6,and CBA mice immunized with synthetic HPV 16 capsids and pooled CFAimmunised control sera.

FIG. 11 illustrates reactivity of two MAbs specific for L1 with a seriesof overlapping peptides of the HPV16 L1 molecule.

FIG. 11A illustrates antigenic index prediction of HPV16L1.

FIG. 12 shows an epitope map of HPV16 L1.

FIG. 13A illustrates synthetic HPV16 VLPs as used for immunisation.

FIG. 13B shows reactivity of the purified VLPs with anti HPV16 L2antiserum by western blot.

FIGS. 14A and 14B show analysis of BPV1 L1 and L2 expression in CV-1cells infected with wild type and recombinant vaccinia viruses; and

FIGS. 15A and 15B show electron microscopy of BPV1 capsids obtained fromcells infected with recombinant vaccinia virus.

EXAMPLE 1 VLPS Derived from HPV16

The HPV-16 L1 gene, from the second ATG (nt5637), was amplified bypolymerase chain reaction from pHPV16 (provided by Dr. L. Gissmann),using following primers:

(SEQ ID NO: 54) 1/ 5′-CAGATCTATGTCTCTTTGGCTGCCTAGTGAGGCC-3′ (SEQ ID NO:55) 2/ 5′-CAGATCTTTACAGCTTACGTTTTTTGCGTTTAGC-3′

The first methionine codon and stop codon are indicated by underline,and BqlII sites were included to facilitate subcloning. The amplified1527 bp fragment was extracted with phenol and purified by 1% agarosegel electrophoresis. After digestion with BglII the L1 gene wassubcloned into the BamHI site of the RK19 plasmid (Kent 1988 Ph.D.thesis, University of Cambridge) which contains a strong vaccinia viruspromoter (4b). The resulting plasmid was sequenced (Sanger et al, 1977,Proc. Natl. Acad. Sci. USA 74, 5463-5467) and used to prepare a fragmentcontaining the HPV16 L1 gene linked to the 4b promoter by digestion withMluI and SstI. This fragment was blunted with T4 DNA polymerase andcloned into the Bam HI site of the vaccinia intermediate vector pLC1,which contains the B24R gene of vaccinia virus (Kotwal and Moss, 1989,J. Virol. 63, 600-606; Smith et al, 1989, J. Gen. Virol. 70, 2333-2343),an E. coli gpt gene (Falkner and Moss, 1988, J. Virol. 64, 1849-1854;Boyle and Coupar, 1988, Gene 65, 123-128) and multiple cloning sites toproduce plasmid pLC200.

The HPV16 L2 gene was prepared by partial digestion of pHPV16 with Acclto produce a fragment (4138 nt-5668 nt) which was filled with Rlenow andlinked to synthetic BamHl linkers. This L2 fragment was cloned into theBam HI site of a pUC derived plasmid termed p480 which has a syntheticvaccinia 28R late promoter, with some modifications (Davison and Moss,1989, J. Mol. Biol. 210, 771-784). The promoter sequence is as follows:

(SEQ ID NO: 56) 5′-GAGCTCTTTTTTTTTTTTTTTTTTTTGGCATATAAATGGAGGTACC C-3′

the late promoter motif is underlined. A fragment containing the L2 genelinked to the 28R promoter was isolated by digestion with SstI/SalI,blunted by T4 DNA polymerase and then cloned into the SstI and SalIsites of pLC200 to produce pLC201 (FIG. 1).

In FIG. 1, HPV16 L1, L2 (open boxes) are under control of vaccinia latepromoters (solid boxes). E. coli gpt gene (shaded box) is used asselection marker. Flanking sequence for homologous recombination. Thedirection of transcription is indicated by arrows.

The pLC201 plasmid was then used to construct a recombinant vacciniavirus as previously described (Mackett et al, 1984, J. Virol. 49,857-864). Recombinant virus pLC201W and pLC202VV were selected by plaqueassay in the presence of mycophenolic acid, xanthine, and hypoxanthine(Falkner and Moss, 1988). Recombinant vaccinia virus (VV) expressingHPV16L1, and HPV16L2, were prepared and used as previously described(Zhou et al, 1990, J. Gen. Virol. 71, 2185-2190).

Recombinant plasmid pLC201 was deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. on 27Mar., 1992 and given the designation 75226.

Recombinant vaccinia virus pLC201VV was deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.on 3 Apr., 1992 and assigned the designation VR2371.

Purification of Virus-Like Particles

CV-1 cells infected with recombinant viruses pLC201VV were harvested in10 mM Tris (pH 9.0) 32 hr after infection and homogenised with a Douncehomogeniser. Homogenates were clarified by centrifugation at 2000 g toremove the cell debris and layered onto a 30% (wt/vol) sucrose cushion.The pellet formed by centrifugation at 110,000 g in a SW38 rotor for 90min was suspended in 10 mM Tris pH9.0 and layered onto a 20-60%discontinuous sucrose gradient. After centrifugation at 100,000 g for 18hrs, 10 equal fractions of 0.25 ml were collected. Samples were mixedwith 0.6 ml ethanol. The pellet obtained after centrifugation at 40 Cand 12000 g for 20 minutes was collected for further analysis. Todetermine the density of the virus-like particles, equilibriumdensity-gradient sedimentation was accomplished in CsCl (1.30 g/ml).After centrifugation at 125,000×g for 20 hrs, 11 fractions of 0.25 mlwere collected. The density of each fraction was determined, and eachwas examined for virus-like particles by transmission electronmicroscopy.

In FIG. 2A cells were infected at 10 pfu/cell with wt VV (lane 1) andpLC201VV (lane 2) and harvested 48 h post infection. L1 proteins wasdetected with the HPV16 L1 specific MAb Camvir 1. The 57 kDa L1 proteinis indicated by the arrow. Binding of Camvirl to the 35 kDa protein inall three lanes is non-specific.

In FIG. 3 RNAs extracted from cells infected with pLC201VV (lane 2),pLC202VV (lane 3) which also incorporates genes encoding HPV16 proteinsE1 and E4 as well as genes encoding HPV16 L1 and L2 or wt VV (lane 4)were resolved on a 1.2% formaldehyde-agarose gel. RNA was transferred tonylon membrane and hybridised with a ³²P-labelled L2 probe.Formaldehyde-treated lambda DNA Hind III cut marker are shown (lane 1).

Electron Microscopy

CV-1 cells infected with recombinant vaccinia virus were fixed in 3%(vol/vol) glutaraldehyde in 0.1M sodium cacodylate buffer and postfixedin 1% osmium tetroxide, dehydrated in graded alcohols, and embedded inepoxy resin. Thin section were cut and stained with uranyl acetate andlead citrate. Fractions from the sucrose gradient were dried onto EMgrids, and negatively stained with 1% (wt/vol) phosphotungstic acid(pH7.0). Fractions were examined using a JEOL 1200Ex Transmissionelectron microscope.

In FIG. 4A there is shown CV-1 cells infected with pLC201W for 32 hours.In the CV-1 nuclei, particles of approximately 40-nm diameter (arrowed)were frequently found. The bar corresponds to 100 nm.

In FIG. 4B there is shown fraction 5 of the sucrose gradient. Papillomavirus-like particles, apparently consisting of regular arrays ofcapsomeres were observed (arrowed). The bar corresponds to 50 nm.

Analysis of HPV ORF Products

HPV16 L1 expression was analysed by immunoprecipitation and immunoblot.For immunoprecipitation, ³⁵S metabolically labelled recombinant VVinfected CV-1 cells were lysed in RIPA buffer 0.1% SDS. 1% Triton X-100,1% sodium deoxycholate, 150 mM NaCl, 0.5 g/ml aprotinin, 10 mM Tris-HCl,pH7.4). Immunoblot analysis of partially purified virus-like particles,using the L1 specific MAb Camvirl (Mclean et al, 1990, J. Clin. Pathol.43, 488-492) and ¹²⁵I antimouse IgG (Amersham), was performed aspreviously described using samples solublised in 2×SDS gel loadingbuffer containing 2-mercaptoethanol. Analysis of HPV16 L2 geneexpression is shown in FIG. 13B. For analysis of N-glycosylation,partially purified virus-like particles were taken up to 100 l buffer(0.25 M sodium acetate, pH6.5, 20 mM EDTA and 10 mM 2-mercaptoethanol)and reacted with 0.5 u Endoglycosidase F (Boehringer Mannheim) at 370 Cfor 18 hrs prior to immunoblotting.

Mycophenolic acid was used to select a vaccinia virus recombinant forthe gnt plasmid pLC201 and this was termed pLC201VV. Synthesis of L1 incells infected with pLC201VV was confirmed by immunoblotting andimmunoprecipation. L1 protein was demonstrated as a band onautoradiography of approximately 57 kDa. A northern blot of RNAextracted from CV-1 cells infected with these recombinant virusesconfirmed high levels of L2 mRNA transcription in cells infected witheither of these viruses (FIG. 3). L2 transcription from a syntheticvaccinia virus late promoter gave a heterogeneous Northern blot patternbecause VV late—RNAs do not use a specific transcription terminationsignal.

CV-1 cells were infected with pLC2O1VV and examined for virus-likeparticles. Electron micrographs of thin sections of cells infected withpLC201VV, but not of control cells infected only with wild-typevaccinia, showed approximately 40 nm virus-like particles in cellnuclei. In most cases these particles were linked in chains, and nearthe nuclear membrane (FIG. 4 a). Cells infected with recombinantvaccinia viruses which expressed HPV16 L1 only or L2 only, and producedthe corresponding protein (L1) or mRNA (L2), did not contain virus-likeparticles. Cells simultaneously infected with two different recombinantvaccinia viruses, which expressed HPV16 L1 and HPV16 L2 respectively,also failed to make any HPV virus-like particles; although L1 proteinand L2 mRNA could be identified in pools of these double infected cellssimultaneous synthesis of both L1 and L2 within individual cells was notdemonstrated.

In FIG. 5 HPV16 virus-like particles obtained from CV-1 cells infectedwith pLC201VV were centrifuged over a sucrose cushion and then subjectedto CsCl isopynic sedimentation. Virus-like particles (+) were found infractions 8 and 9. The transmission electron micrograph of a negativelystained particle from fraction 8 is shown in the insert. The density(g/ml) of each gradient fraction is indicated.

In FIG. 6 CV-1 cells were infected with pLC201VV for 32 hrs, andvirus-like particles were purified on a sucrose gradient. Samples wereprecipitated with ethanol and treated with endoglycosidase F beforeanalysis by immunoblotting with an anti-HPV16 L1 antibody Camvir I. Lane1: purified virus-like particles; Lane 2 and 3: after treatment withendoglycosidase F overnight. The L1 doublet is indicated by (=), anddeglycosylated L1 is indicated by the arrow. Molecular weight markersare shown on the left.

To confirm that the virus-like particles observed by electron microscopycontained HPV16 L1 protein, cell extracts from pLC2O1VV infected cellswere subjected to a partial purification in a 20%-60% sucrose gradient.Ten fractions were collected and examined for L1 protein. From fractions3 to 7, L1 could be detected and in fraction 5, the highest level of L1was found. Each fraction was also examined by EM for virus-likeparticles: these were observed in fraction 5. A typical papilloma virusnegatively-stained with sodium phosphotungstate, has 72 regularclose-packed capsomeres (Finch and Klug, 1965, J. Mol. Biol. 13, 1-12;Rowson and Mahy, 1967, Bacteriol. Rev. 31, 110-131) and has a diameterabout 50 nm. The diameter of the virus-like particles purified from theinfected CV-1 cells varied between 35 nm and 40 nm. These virus-likeparticles however possessed a similar EM appearance to papillomaviruses, and a regular array of capsomeres could be recognised (FIG. 4b). The virus-like particles identified in fraction 5 of the sucrosegradient were therefore presumed to be empty and incorrectly assembledarrays of HPV capsomeres. In CsCl, HPV16 virus-like particles sedimentedat about 1.31 g/ml (FIG. 5), and showed a typical empty papilloma viruscapsid appearance under transmission electromicroscope (FIG. 5, insert).

Canvir-1 identified a protein doublet in western blots of virus-likeparticles purified from pLC20iVV infected CV-1 cells (FIG. 6). HPV16 L1contains four potential N-glycosylation sites (asparagine 157, 242, 367and 421). To test whether the doublet represented glycosylation variantsof the L1 polypeptide, partially purified virus-like particles weresubjected to treatment with endoglycosidase F, prior to SDS-PAGE andimmunoblotting. This resulted in the replacement of the doublet by asingle band of slightly lower apparent molecular weight, at the expectedmolecular weight of about 57 kDa (FIG. 6. lane 2, 3).

The virus-like particles collected from fractions of the CsCl gradientwith a buoyant density of 1.29-1.30 g/ml were used as antigen in anELISA assay. All antisera from mice immunised with VLPs were positive(Table 2). Control sera from mice immunized with the similar fractionsof a density gradient prepared with lysate of CV-1 cells infected withwild type vaccinia were nonreactive with the virus-like particles. Usingtwo different protocols to coat virus-like particles to ELISA plates(Dillner et al., 1991, J Virol 65, 68626871), attempts were made todistinguish reactivity with native HPV virus-like particles fromreactivity with the partially denatured proteins of disrupted particles.The murine antisera raised against the VLPs were equally reactive withthe native (OD 1.00+0.20) and denatured (OD 1.60+0.45) particles. Apanel of 6 monoclonal antibodies specific for defined L1 epitopesincluded only 1 (Camvir 1) that was weakly reactive (OD 0.064) withnative VLPs, and it proved more reactive with denatured particles (OD0.107) than with native particles, suggesting that the reactivity waswith denatured L1 protein in the native VLP preparation.

In FIG. 8 ¹²⁵I-labelled anti-mouse IgG was used as the second antibody.The 57-kDa L1 band is indicated by the arrow. Key: sera from individualmice immunized with VLPs: lanes 1-5, BALB/c; lanes 6-10, C57B1/6; lanes11-15, B1OA; sera from individual mice immunized with CFA; lane 16,BALB/c; lane 17, C57B1/6; lane 18, B1OA; anti-HPV 16L1 MAb Camvir 1,lane 19. The molecular weights are indicated on the left.

Reactivity of the anti-VLP antisera with the L1 and L2 proteins of HPV16was confirmed by immunoblot using baculovirus recombinant HPV16 L1 andL2 proteins. Sera from all mice immunized with the virus-like particles,and each of the monoclonal anti-HPV 16 L1 antibodies, recognized a57-kDa protein (FIG. 8) in the L1 recombinant-baculovirus-infected S.frugiperda cell lysates, and no comparable reactivity was observed withlysates of S. frugiperda cells infected with wild-type baculovirus. Theintensity of reactivity with the L1 protein varied from mouse to mouse,but all sera were reactive with prolonged exposure of the immunoblots.Similar results were obtained with the L2 recombinantbaculovirus-infected cell lysates: murine anti-VLP antisera and a rabbitantiserum to L2 protein both reacted with a single protein in thelysate. Sera from mice immunized with CFA alone failed to react withprotein from lysates of L1 or L2 recombinant baculovirus-infected S.frugiperda.

EXAMPLE 2 Definition of Linear Antigenic Regions of HPV16L1 ProteinUsing VLPS

In a further series of experiments the linear antigenic regions of theHPV16 L1 capsid protein using synthetic VLPs were determined. In suchexperiments mice of three haplotypes (H-2^(d), H-2^(b), and H-2^(d/b))were immunized with synthetic HPV16 virus-like particles (VLPs),produced using a vaccinia virus doubly recombinant for the L1 and L2proteins of HPV16. The resultant-anti-VLP antisera recognized HPV16capsids by ELISA assay and baculovirus recombinant HPV16L1 and L2protein on immunoblot. Overlapping peptides corresponding to the HPV16L1amino acid sequence were used to define the immunoreactive regions ofthe L1 protein. The majority of the L1 peptides were reactive with IgGfrom the mice immunized with the synthetic HPV16 capsids. A computeralgorithm predicted seven B epitopes in HPV16 L1, five of which laywithin peptides strongly reactive with the murine antisera. The murineanti-VLP antisera failed to react with the two peptides recognized byanti-HPV16L1 monoclonal antibodies raised by others against recombinantL1 fusion protein. We conclude that the immunoreactive epitopes of HPV16defined using virus-like particles differ significantly from thosedefined using recombinant HPV16L1 fusion proteins, which implies thatsuch fusion proteins may not be the antigens to look for HPV16L1specific immune responses in HPV-infected patients.

Production of HPV16 capsids. Plasmid pLC201 containing HPV16L1 and L2open reading frames (ORFs) under the control of vaccinia virus promoters4b (natural) and p480 (synthetic) was used to construct the recombinantvaccinia virus (rVV) pLC2O1VV as previously described but withexceptions as mentioned below. HPV16 virus-like particles were preparedfrom pLC201VV infected CV-1 cells as mentioned previously, but cellswere cultured in medium containing rifampicin at 100 g/ml to prevent theassembly and maturation of vaccinia virus (Moss, “Virology” p685-703Raven, New York, 1985; Raracostas et al., PNAS 86, 8964-8967, 1989). Theinfected cells were harvested and lysed by freezing and thawingfollowing Dounce homogenization in 10 mM Tris-HCl (pH 9.0). Lysates wereclarified by centrifugation at 2000 g and then spun at 100,000 g for 2hr over a 20% sucrose cushion in PBS buffer. The pellet was mixed withCsCl to an initial density of 1.30 g/ml and centrifuged at 100,000 g for18 hr at 18°. Fractions were collected and immunoblots were performed onethanol-precipitated proteins. Fractions testing positive for L1 proteinwere pooled, and the presence of virus-like particles confirmed byelectron microscopy as described above.

Production of antisera. Groups of five mice BALB/c (H-2^(d)), C57B1/6(H-2^(b)), and B1OA (H-2^(b/d)) were immunized with CsClgradient-purified HPV16 virus-like particles. Animals were inoculatedwith 5 g of capsid protein by subcutaneous injection. The initialinjection was given with Freund's complete adjuvant, and three furtherinjections at 3 weeks' intervals were given in saline. Fourteen daysafter the fourth injection, sera were collected and stored at −20°.Material prepared from CV-1 cells infected with wild type vacciniavirus, and processed exactly as for pLC201VV infected cells, was used toimmunize control groups of mice according to the same protocol.

Peptides. A series of 15-mer peptides, overlapping by five residues, andspanning the deduced amino acid sequence of HPV16L1 protein (Seedorf etal., 1985, Virology 145 181-185; Parton, 1990, Nucleic Acids Res 18 363)was synthesized with the DuPont RaMPS multiple peptide synthesis systemusing Fmoc chemistry according to standard protocols (Fields and Noble,1990 Int. J. Pept. Protein Res 35 161-214) and then conjugated withglutaraldehyde to bovine serum albumin (BSA). To denote the position ofthe amino acids (aas) in the L1 protein, the putative first initiationcodon was designated amino acid number 1 (Table 1). For technicalreasons the C-terminal peptide corresponding to aas 521531 was not used.All peptides used were of greater than 85% purity as judged by HPLCanalysis.

Recombinant L1+L2 proteins. Recombinant baculoviruses expressing theHPV16L1 or the HPV16L2 ORF were used to infect insect SF9 cells. After 3days incubation at 25°, cells were pelleted by centrifugation at 14,000g for 5 min. The pellet was dissolved in RIPA buffer (20 mM Tris-HCl, pH7.6; 2 mM EDTA; 50 mM NaCl; 1% deoxycholate; 1% Triton X-100; 0.25% SDS;1% aproptinin; 1 mM PMSF).

Western blotting. Virus like particles or recombinant L1 or L2 proteinwere mixed with 2× loading buffer containing 2% SDS/DTT and boiled for 5min. The proteins were separated in 10% polyacrylamide gels and blottedonto nitrocellulose (Towbin et al., 1979, Virology 175 1-9). Filterswere cut into strips, incubated in 3% BSA in PBS at 37° for 1 hr.Blocked strips were exposed to the various murine antisera (1:200) ormonoclonal antibodies overnight at 40. The reactive proteins werevisualized by autoradiography after reaction with ¹²⁵I-conjugatedanti-mouse IgG (0.2 Ci/ml) (Amersham).

ELISA assay. Polyclonal antisera were tested for reactivity withsynthetic HPV16 capsids by an enzyme linked immunosorbent assay (ELISA)as previously described (Christensen et al., 1990, PNAS 76 4350-4354,Cowsert et al., 1987, JNC1 79 1053-1057). For assays with “native”synthetic HPV16 capsids, 100 ng of protein in PBS (pH 7.5) was attachedto each ELISA plate well (Flow Labs) by incubation for 1 hr at 37°. Forassays with “denatured” particles, the particles were suspended incarbonate buffer, pH 9.6, and adsorbed on to the plate overnight at 37°.All subsequent incubations were done at room temperature. The plateswere washed with PBS, and unattached sites were blocked by incubationfor 1 hr in blocking buffer (5% milk powder in PBS, pH 7.5). The murineantisera (1:200), previously absorbed with wildtype VV-infected CV-1cell extract, were added and incubated for 1 hr, and the plates werewashed with PBS. Horseradish peroxidase-conjugated anti-mouse IgG(Sigma) at 1:1000 dilution in blocking buffer was added and incubatedfor 1 hr, followed by 10 washes with PBS. Substrate buffer (pH 4.6)containing ABTS (Boehringer) and H₂O₂ was added and the OD415 read after15 min.

Linear B epitope mapping. B epitopes were identified by screeningantisera from immunized animals against the set of overlapping HPV16L1peptides by ELISA. Synthetic peptides coupled to BSA were diluted in 10mM sodium carbonate buffer (pH 9.3) and adsorbed to ELISA platesovernight at 4°. Blocking of residual binding sites on the plates wascarried out using 3% BSA in PBS for 2 hr at 37° Diluted mouse antisera(1:500) were incubated with coated plates at room temperature for 2 hr.The plates were washed with PBS containing Tween 20 (0.1%) and incubatedwith peroxidase-conjugated antimouse IgG (1:1000) (Sigma) or IgA(1:2000)(Sigma) for 2 hr. Plates were washed and developed with 0.5mg/ml ABTS in substrate buffer (pH 4.6) for 15 min before recordingabsorbance values at 415 nm. A peptide was considered reactive if the OD415 value with the test serum was greater than 3 SDs above the mean forthe control serum: this gave a cut-off value of 0.260. An OD 415 of fivetimes the mean OD 415 obtained with control sera (0.55) was arbitrarilyconsidered to define a major reactive epitope.

Monoclonal antibodies and antisera. Five monoclonal antibodies (MAb)raised against HPV16L1 fusion protein were used. MAb 5A4, ID6, 3D1, and8C4 (Cason et al., 1989, J. Gen Virol 70 2973-2987) were provided by Dr.Phil Shepherd from London, U.R. and MAb Camvir 1 (McLean et al., 1990,J. Clin. Pathol. 43 488-492) was obtained from Dr. C. McLean (Departmentof Pathology, University of Cambridge). Rabbit antiserum to HPV16 L2

Trp-E fusion protein was provided by Dr. Denise Galloway (University ofWashington, Seattle).

Amino acid sequence analysis and the antigen index prediction. Theantigenic index (AI) (Jameson and Wolf, 1988, Comp. Appl., Biosci 4181-186) is a measure of the probability that a peptide sequence isantigenic. It is calculated by summing several weighted measures ofsecondary structure. Values for the predicted HPV16L1 sequence werecalculated using PLOTSTRUCTURE software.

In FIGS. 9 and 10, reactivity (OD 415) of the sera in ELISA with aseries of overlapping peptides corresponding to the sequence of HPV16 L1is shown. Peptide numbers corresponding to the HPV16L1 sequence (seeTable 1) are indicated.

In FIGS. 11 and 11A, the numbering system for the amino acids correspondto HPV16L1 from the first putative initiation codon. The regions with AIvalue over 1.5 are indicated.

In FIG. 12, the regions of HPV16L1 within which B epitopes have beenshown to lie in a range of mapping systems are shown. Results with serafrom mice immunized with the VLPs (Particles) and with IgA and IgGantibodies in sera from humans with cervical cancer (Human IgA and HumanIgG) (Dillner et al. 1990 Int. J. Cancer 45 529-535) were obtained usingoverlapping peptides. The murine anti VLP antisera were held to besignificantly reactive with a peptide if the OD was greater than 0.55.Results from rabbits immunized with an L1 fusion protein (Rabbit Serum)(Muller et al. 1990 J Gen Virol 71 2709-2717) are plotted: these weredetermined using a series of partial-length expression clones and thewhole length of the sequence within which the epitope(s) lay is shown.Also indicated are the algorithm-predicted B epitopes (Computer)(Jameson and Wolf Comp. Appl. Biosci 4 181-186 1988) and the epitopesrecognised by the published anti-HPV16L1 monoclonal antibodies(Monoclonals) (Cason et al. J. Gen Virol. 70 2973-2987 1989; McLean etal. J. Clin Pathol. 43 488-492 1990). The scale shows the position ofthe epitopes along the 531 aa L1 protein, and is numbered from theN-terminal methionine (residue 1). For each epitope containing peptide,the exact location with regard to the N-terminal methionine is alsogiven.

A series of 15-mer synthetic peptides of the HPV16 L1 protein, coveringthe whole length of the protein with five aa overlaps, was used todefine the epitopes in L1 recognized by the various immune sera. Each of16 antisera from the three tested inbred mouse strains. BALB/c(H-2^(d)),C57B1/6(H-2^(b)), and B1OA (H-2^(b/d)), recognized multiple linearpeptides of the L1 protein, and essentially the same peptides from HPV16L1 were recognized by all strains tested (FIG. 10). Five individual serawere tested from each strain. A peptide was designated reactive if theOD with a serum was greater than the mean+3SD of the ODs of the negativecontrol sera; this gave a cutoff for reactivity of 0.260. Sera from miceimmunized with CFA alone had an OD 415 reactivity of less than 0.260with all the L1 peptides. While some variation was seen in the intensityof the reactivity of each anti-VLP serum from a given strain with eachpeptide, each of the peptides was reactive (OD>0.260) with either all ornone of the anti-VLP sera from each strain of mouse. The isotype of thepeptide specific antibody in the anti-VLP sera was examined using IgG-and IgA-specific anti-mouse immunoglobulin antibodies. The IgG responsewas as shown (FIGS. 9 and 10) and no significant IgA reactivity could bedetected to any peptide (OD<0.050). As most peptides were reactive withthe anti-VLP sera an arbitrary OD value of five times the mean negativevalue was used to define major reactive regions of the L1 protein: themajor immunoreactive L1 peptides were evenly distributed along thelength of the L1 protein, as seven were in the amino terminal third ofthe molecule, seven in the middle third, and eight in the carboxyterminal third. In contrast, the monoclonal antibodies specific forHPV16 L1 recognized single major linear epitopes as previously described(Cason et al., above, McLean et al, 1990 above). Four of the fivereactive anti-HPV16L1 monoclonal antibodies (5A4, ID6, 3D1, 8C4) werereactive with peptide 30 (291-305), whereas Camvir 1 recognized peptide24 (231-245) (FIG. 11 and ill). Sera from mice immunized with thevirus-like particles failed to react with either of these peptides.

An algorithm was used to deduce likely B epitopes of HPV16L1, based onthe predicted protein secondary structure. Possible antigenic regionswere calculated as an antigenic index (AI) (Jameson and Wolf, 1988,above) on the basis of chain flexibility, high accessibility and highdegree of hydrophilicity (FIG. 11). A region with an Al value over 1.5was regarded as a predicted B epitope. Seven such regions were found(amino acids 79-84, 105-108, 120-122, 134-135, 267, 269, 298-299,363-367) and five of these seven regions were within the 22 peptides towhich major reactivity was seen with antisera from mice immunized withsynthetic HPV16 capsids. The summary of the B epitope specificity ofantisera from different sources is shown in FIG. 12.

In further consideration of Examples 1-2 it is noted that papillomaviruses generally produce virions in infected keratinocytes which arereadily identifiable by electron microscopy (Almeida et al, 1962, J.Invest, Dermatol 38, 337-345) and which in some cases can be purifiedand shown to be infectious (Rowson and Mahy, 1967, Bacterial. Reo. 31,110-131). HPV 16 virions are however, not seen in HPV16 infectedcervical epithelial tissue although HPV16 L1 and L2 late genetranscription occurs in differentiated genital epithelium (Crum et al,1988. J. Virol. 62, 84-90) and L1 translation produces immunoreactive L1protein in these tissues (Stanley et al 1989, Int. J. Cancer 43,672-676). In this specification we have shown that expression of HPV16L1 and L2 genes in epithelial cells is both necessary and sufficient toallow assembly of HPV16 virion-like particles and thus the L1 and L2proteins of HPV 16 are not defective with regard to virion assembly. Theexpression of HPV16 late genes in tissues appears to be strictlyregulated by the epithelial environment (Taichman et al, 1983, J.Invest. Dermatol 1, 137-140). Failure to detect HPV16 virions in vivo,despite transcription of L1 and L2 and translation of L1, suggests thatthere is either a post transcriptional block to L2 production incervical epithelium, or an inhibitor of virion assembly. In the HPV16containing cell line W12, derived from cervical tissue, virus-likeparticles were observed when the cells underwent terminaldifferentiation in vivo in a murine microenvironment (Sterling et al1990. J. Virol 64, 63056307) suggesting that such cells have no block tovirion assembly, and that insufficient translation of L2 or otherunknown reasons may explain failure to demonstrate HPV16 virions incervical tissues.

Our EM studies show that the empty HPV16 virion has an average size ofabout 40 mm which is smaller than other papilloma viruses, but has asimilar surface structure compared with other papilloma viruses such asrabbit papilloma virus (Finch and Klug, 1965 J. Mol. Biol. 13 1-12), orhuman wart virus (Rowson and Mahy 1967 above). Sedimentation showed anempty capsid density of about 1.31 g/ml, the density expected of emptypapilloma virus capsid compared with about 1.36 g/ml for intact HPV1avirions (Doorbar and Gallimore, 1987, J. Virol. 61, 2793-2799).

The L1 protein from HPV has potential glycosylation sites, and purifiedBPV particles have minor electrophoretic forms of L1 whose mobility issensitive to endoglycosidase treatment (Larsen et al, 1987, J. Virol 61,3596-3601). L2 from HPV 1a and HPV 11 has been observed to be a doublet(Rose et al, 1990, J. Gen. Virol, 71, 2725-2729; Doorbar and Gallimore,1987 above; Jin et al, 1989, J. Gen. Virol. 70, 1133-1140) and this hasbeen attributed to differences in glycosylation. Our data show that theL1 protein in HPV16 capsomeres is also glycosylated, and that twodifferent glycosylation states exist.

In this specification we used synthetic virus like particles to studyimmunogenicity of the HPV16 capsid proteins produced in a eukaryoticsystem. Capsid proteins produced in eukaryotic cells were used sincepapilloma-virus capsid proteins produced in eukaryotic cells undergopost-translational modification (Browne et al., 1988 J. Gen. Virol. 691263-1273; Zhou et al. 1991 Virology 185 625-632) which may be animportant determinant of antigen presentation. A recombinant vacciniaexpression vector was chosen because no native HPV16 particles areavailable from clinical lesions, or from viral propagation in cellculture. We used the HPV16 VLPs to produce polyclonal anti-VLP antiserain mice, and these sera reacted strongly with the HPV16 capsids byELISA. We have demonstrated by immunoblotting that the anti-VLP antiserarecognized epitopes in denatured L1 (FIG. 2) and L2. Moreover, anti-VLPsera defined 22 major reactive peptides in a series of fifty one 15-merpeptides of L1. These data indicate that antisera raised against viralparticles nevertheless frequently recognize linear determinants. Theprofile of humoral reactivity with the set of L1 peptides was almostidentical across two MHC disparate mouse strains, suggesting that thereare sufficient T epitopes in L1 that MHC restriction is not limiting indetermining the humoral response to the HPV16 L1 protein in the mousestrains tested here.

The data for B epitope specificity obtained with our murine anti-VLPantisera can be compared (FIG. 12) with a similar study of “immune”serum from women with cervical dysplasia (Dillner et al., 1990 Int. JCancer 45 529-535). Several peptides were recognized by both immunehuman sera and the anti-VLP antisera, but the majority of peptidesreactive with the murine anti-VLP antisera were not reactive with theimmune human sera (FIG. 12). Neither of the regions of L1 (221-235,291305) recognized by L1-specific monoclonal antibodies (Cason et al.,1989; McLean et al., 1990) were recognized by our murine anti-VLPantisera. As L1 fusion proteins were used to raise these MAbs, and havealso been used to screen for antibody to L1 in human serum, the lack ofreactivity of human sera with L1 fusion protein (Jenison et al., 1990 J.Infect. Dis 162 60-69; Söchel et al., 1991 Int. J. Cancer 48 682-688)may be explained by the failure of the L1 fusion proteins to display theepitopes of L1 which are presented to the human immune system by nativeL1 protein.

Screening for antibodies to the L1 Protein with peptides can detect onlylinear epitopes. In an attempt to determine whether the reactivity inthe murine sera was directed against both linear and conformationaldeterminants we carried out ELISA assays with the particles treated intwo ways: one said to preserve native particles and the other to producedenatured protein (Dillner et al., 1991 J. Virol. 65 6862-6871). We didnot fine any serum or monoclonal antibody reactive exclusively withparticles treated in one or other manner, though one MAB (Camvir 1)reacted more strongly with the denatured that the “native” particles.Lack of reactivity of the majority of the MAbs with the denaturedparticles suggests that they were only partially denatured, as the sameantibodies react with denatured protein in a Western blot. Conversely,the reactivity of Camvir 1 with the native particles is not proof thatthe linear epitope recognized by this antibody is recognizing denaturedL1 protein present in some amount in the native particle preparation,and we have no proof that intact VLPs are preserved under our ELISAconditions.

Since most antibodies recognize conformation dependent determinants(Benjamin et al., 1984 Ann. Rev. Immunol. 2, 67-101), which can involveseveral noncontiguous polypeptide sequences (Amit et al., 1986 Science233 747-753), antibodies elicited to virions are unlikely to recognisefused or denatured proteins as well as the native protein, as has beenshown for HPV1 antisera (Steele and Gallimore, 1990 Virology 174388398). Virions of some skin-wart-associated HPV are available inquantities sufficient for serological assays (Almeida and Goffe, 1965Lancet 2 1205-1207; Kienzler et al., 1983 Br J. Dermatol. 108 665-672;Pfister and Zur Hausen, 1978 Int. J. Cancer 21 161-165; Pyrhsonen etal., 1980 Br. J. Dermatol 102 247-254; Pass and Maizel, 1973 J. Invest.Dermatol 60 307-311) for wart parings. The prevalence of antibodies topurified virions in human immune serum varies from 20 (Genner, 1971Acta. Derm. Venereol (Stockh) 51 365-373) to 88% (Morison, 1975 Br JDermatol 93 545-552) depending on the detection system used. However,until recently, virions of the genital HPV types have been unavailablefor serological study. The nude mice xenograft system (Kreider et al.,1987 J Virol 61 590-593) has allowed production of HPV11 particles forthe detection of human antibodies (Bonnez et al., 1991 J. Gen Virol 721343-1347). We anticipate that the HPV16 VLPs described here will allowsimilar studies of seroreactivity to native HPV16 particles to bedeveloped, and the observed lack of reactivity in human serum to HPV15L1fusion proteins (Jenison et al., 1991 J Virol 65 1208-1218; Kochel etal., 1991 above) may simply parallel the similar observations with HPV1(Steele and Gallimore, 1990 Virology 174 388-398).

Antibodies to BPV structural proteins have virus-neutralizing activity(Pilacinski et al., 1986 Ciba Found. Symp. 120 136-156) and antiseraraised against purified HPV11 virions could also neutralize infectiousHPV11 in an athymic mouse xenograft system (Christensen and Kreider,1990 J Virol 64 3151-3156). Our results indicated that the purifiedsynthetic HPV16 capsids are immunogenic and could be used to produce andevaluate virus-neutralizing antibodies specific for this oncogenicvirus. BPV1 L1 protein expressed in Escherichia coli and BPV particleshave both protected cattle from development of warts (Pilacinski et al.,1986; Jarrett et al., 1990 Vet. Rec. 126 449-452). A similar immuneresponse to HPV16 virus-like particles would be the basis of a potentialvaccine to prevent HPV16-associated cervical cancer.

In FIG. 13A, after infection with pLC201VV, CV1 cell lysates weresubjected to equilibrium gradient sedimentation. Purified virus-likeparticles were examined by transmission electron microscopy afternegative staining with Phosphotungstic acid. [bar=100 nm].

In FIG. 13B, Lane 1, the lysate from CV1 cells infected with wild typevirus; lane 2, lysate from L1 and L2 expressing virus pLC201VV; lane 3,purified virus-like particles. The L2 protein was probed with a rabbitanti HPV16 L2 antibody followed by 1251-protein A. The molecular weightsare indicated on the left and L2 bands are arrowed.

Reference may also be made to FIGS. 13A and 13B which show that HPV16 L1and L2 double recombinant W contain L2 protein as demonstrated bywestern blot, using purified VLPs and a rabbit antiserum raised againstVV recombinant L2 protein.

EXAMPLE 3 BPV1 VLPS

It has also been ascertained that bovine papillomavirus (BPV) 1 virionssimilarly produced in vitro using W recombinant for the BPV1 capsidproteins can package BPV1 DNA. Complete virions are able to infect apermissible mouse fibroblast cell line, as indicated by transcription ofthe E1 viral open reading frame, and infection is inhibited byincubation of virions with antibodies to the capsid protein of BPV1. Incontrast to the observations for HPV16, virus like particles assemble incells infected with W recombinant for the BPV1 L1 capsid protein alone,but L2 protein is required to package BPV1 DNA to produce infectiousvirions.

With reference to the HPV16 VLPs referred to above, these particlesappeared to consist of capsomeres typical of those seen in HPV1 and BPV1particles purified from clinical lesions (Bakar et al, J. C. Biphys J 601445-1456-1991, Staquet et al., J. Dermatologica 162 213219, 1981),though the overall morphology of the HPV16 particles was ratherdifferent to naturally occurring HPV1 and BPV1 particles. As naturalHPV16 virions have not been purified from clinical lesions, it wasconsidered desirable to ascertain whether this morphological differencewas a property of HPV16, or the recombinant vaccinia virus (rVV) systemused to produce the virions. A series of VVs were therefore made, eachdoubly recombinant for the L1 and L2 caps id proteins of HPV6, HPV11,and of BPV1. Infection of CV-1 cells with each of these doublerecombinant VVs produced virus like particles, and these resembled theauthentic HPV1 and BPV1 virions more closely than the HPV16 particles.We chose to study the BPV-1 particles, as natural BPV-1 particles arebetter characterised morphologically and immunologically (Chen et al,Baker et al 1991, Cowsert et al., J. Natl Cancer Inst. 79 1053-1057) andcell lines are available which are permissive for the episomalreplication of BPV-1 DNA (Law et al 1981 DNAS 78 27272731).

In this example, BPV1L1 is expressed from the p4b natural vaccinia latepromoter and L2 from the p480 synthetic vaccinia late promoter. The E.coli gpt gene is used as the selection marker. Flanking sequences arethe vaccinia B24k gene, which provides a vaccinia sequence forhomologous recombination. The BPV1 L1 and L2 genes were cloned by PCRfrom plasmid pml-1. Because the BPV1 genome is linearised and clonedinto this plasmid at a BamHI site in the BPV1 L2 ORF, the BPV1 genomewas first isolated from pml-1 by BamHI digestion and recircularised, andthe circularised BPV-1 DNA was used as the PCR template. Oligonucleotideprimers used for L1 amplification were:

(SEQ ID NO: 57) 5′-CGGGATCCATGGCGTTGTGGCAACAAGGCCAGAAGCTG. (SEQ ID NO:58) 5′ CGGGATCCTTATTTTTTTTTTTTTTTTGCAGGCTTACTGG.

The BamHI site is underlined and the first methionine and stop codonsare in bold.

Oligonucleotide primers for L2 amplification were:

(SEQ ID NO: 59) 5′-GCAGATCTATGTGCACGAAAAAGAGTAAAACGTGCCAGTGC. (SEQ IDNO: 60) 5′ GCAGATCTTTAGGCATGTTTCCGTTTTTTTCGTTTCC.

The Bgl II sites are underlined and the first methionine and stop codonsare in bold. The amplified 1478 bp L1 fragment was cloned into the BamHIsite in plasmid RK19 to produce RK19BPVL1. The L1 gene and vaccinia 4bpromoter were isolated from this plasmid by digestion with MluI and SmaIand transferred into plasmid pSX3 to produce pSXBPVL1. The 1409 bp L2fragment was digested with Bgl II and cloned into the BamHI site inplasmid p480 to produce p480BPVL2. The synthetic vaccinia late promoterand BPV L2 gene were cloned from this plasmid into the Sma1 site inpSXBPVL1 to produce the doubly recombinant plasmid pSXBPVL1L2.Transfection of pSXBPVL1 or pSXBPVL1L2 DNA into monolayers of CV-1infected with wild type (wt) VV WR strain resulted in the rVVs pSXBPVL1W(L1 expressing) and pSXBPVL1L2VV (L1 and L2 expressing). Recombinantvaccinia viruses were purified three times in presence of mycophenolicacid. Following purification, large-scale preparations of therecombinants were made and used throughout these experiments.

In FIG. 14A there is shown immunoprecipitation analysis of recombinantBPV1 L1 in vaccinia-infected cells. CV-1 cells were infected at 10pfu/cell with wt vaccinia virus (Lane 1); pSXBPVL1VV (lane 2); pSXBPVL1L2W (lane 3) and harvested 48 hrs postinfection. BPV1L1 protein wasdetected with BPV1 specific rabbit antiserum. The 58 kDa L1 protein isindicated by an arrow.

In FIG. 14B there is shown northern blot analysis of BPV1 L2 expression.RNA extracted from CV-1 cells infected with wt W (lane 1); pSXBPVL1L2VV(lane 2) was probed with the BPV1 L2 gene. The variable length of the L2homologous transcripts is typical of transcripts expressed from Wpromoters. For immunoprecipitation, cells infected with rws were lysedwith RIPA buffer (0.1% SDS, 1% Triton X-100, 1% sodium deoxcholate, 150mM NaCl, 0.5 g/ml aprotinin, 10 mM Tri.s, pH7.4). Soluble proteins wereimmunoprecipitated with rabbit anti BPV1 antibody (DAKO, Glostrup) at1:1000 dilution. Precipitated proteins were collected with protein-Asepharose, separated on 10% SDS polyacrylamide gels and blotted ontonitrocellulose filters. After blocking with skim milk, the filters wereexposed to the anti BPV1 serum (1:1000) followed by 1251-protein A (0.1pCi/ml) and visualised by autoradiography. Total RNA was extracted fromcells, and purified by centrifugation through CsCl as describedpreviously. Total RNA, 30 g per track, was run on 1.2%formadelhyde-agarose gels, transferred to nylon membranes, and probedwith a 32P-labelled BPV1 L1 gene.

In FIG. 15A reference is made to pSXBPVLiL2VV. Some particles fromCON/BPV cells infected with pSXBPVL1L2W have electron dense cores(Insert).

In FIG. 15B reference is made to pSXBPVL1W. A contaminating vacciniavirus in (A) is indicated by a “V”. The scale bars represent 50 nm.Cells were harvested two days postinfection, washed with PBS, lysed byfreeze and thaw three times and sonicated in PBS. Cell debris wasremoved by low-speed centrifugation, and the supernants were layered ona 20% (w/v) sucrose cushion and centrifuged for 2 hours at 100,000×g.The pellets were resuspended in PBS and analysed in a JEOL 1200EXelectron microscope after negative staining with 1% (w/v)phosphotungstic acid (pH 7.0).

As the ratio of the L1 to L2 proteins in authentic BPV1 particles isapproximately 5:1 (Pfister, H. & Fuchs, E. in Papillomaviruses and humandisease (eds Syrjanen, K., Gissman, L & Koss, L. G.) Vol. 1-18(Springer-Verlag, Berlin, 1987), we used a strong natural promoter forthe L1 gene and a weak synthetic promoter for the L2 gene for our doublyrecombinant VV, and the resulting ratio of L1 mRNA to L2 mRNA on anorthern blot from rVV infected CV-1 cells was approximately 10:1. CV-1cells infected with this rW expressed BPV1 L1 protein (FIG. 14A) and L2mRNA (FIG. 14B), and large numbers of 50 nm icosahedral virus-likeparticles of apparently authentic morphology (FIG. 5 a) could bepurified from the infected cells. Our previous work with HPV16 had shownthat both L1 and L2 proteins were required for the assembly of HPV 16virus-like particles as described above, which contrasts with theobservation that, for parvovirus (Rajigaya et al, 1991 DNAS 46464650),bluetongue virus (Loudon et al, 1991 Virology 182, 793-801), and polyomavirus (Salunke et al, 1986, Cell 46, 895-904), virus like particlesassemble in cells if the major capsid protein alone is expressed as arecombinant protein. We therefore produced a VV recombinant for BPV1 L1and observed that when CV-1 cells were infected with this VV, virus likeparticles of similar morphology to those obtained with the L1 and L2double recombinant W could be purified (FIG. 15 b). Similar emptyicosahedral virions were obtained after infection of CV-1 cells with VVrecombinant for the L1 proteins of HPV6 or HPV11. The lack ofmorphologically authentic PV virion in cells infected with the HPV16L1and L2rW, and the lack of PV virions seen in HPV16 infected tissue byelectron microscopy (Schneider (1987) Papillomaviruses and Human DiseaseVol 19-39 Springer Verlag Berlin), suggests that HPV16 in contrast toother PVs may be defective with respect to viral capsid formation.

A minority of virus like particles from cells infected with the VPV1L1/L2 rVV is shown in the FIG. 15 a insert.

The cloning strategy for HPV11L1/L2 and HPV6bL1/L2 double expressingrecombinant vaccinia viruses is described below:

For HPV11L1:

(SEQ ID NO: 61) 5′ CAGATCTCAGATGTGGCGGCCTAGCGACAGCACAGTATATGTGCC (SEQ IDNO: 62) 5′ CGGGAATTCGTGTAACAGGACACACATAATAATTGTTTATTGCACAA AA

The PCR product was digested by BgIII/EcoRI and cloned into RK19 undercontrol of 4b promoter. The promoter/11L1 sequence was then cloned intopSX3 BamHl site blunted by Klenow. The resultant plasmid was pSX11L1.

For 11L2

(SEQ ID NO: 63) 5′GCGGATCCATGAAACCTAGGGCACGCAGACGTAAACGTGCG (SEQ ID NO:64) 5′CGCCCGGGCTAGGCCGCCACATCTGTAAAAAATAAGGG

The Bam/HI/Smal digested PCR fragment was cloned into p480 undersynthetic 28 k late promoter. Then the promoter/11L2 fragment wastransferred to pSX11Li. The 11L1/L2 double recombinant expressingplasmid was named as pSX1iL1/L2.

For HPV6bll:

(SEQ ID NO: 65) 5′CGCCCGGGTTACCTTTTAGTTTTGGCGCGCTTACGTTTAGG (SEQ ID NO:66) 5′GCGGATCCAGATGTGGCGGCCTAGFCGACAGCACAGTATATG

The PCR product was cut by BamH1/Sma1 and cloned into RK19 under controlof 4b promoter. The promoter/6L1 were then cloned into pSX3 to producepSX6L1.

For HPV8L2:

The HPV6L2 was isolated from 6b genome by Accl/Xbal (4422-5903). Thefragment was blotted by klenow and inserted into p480. The synthetic 28k promoter plus 6bL2 was cloned into pSX6L1 to form double recombinantplasmid pSX6L1/L2.

Thereafter plasmids pSX11L1 and pSX1iL1/L2 infected a host cell (eg CV1cells or C127 cells) to produce VV p SX11L1 and VV pSX11 L1/L2 whichafter transfection of a host cell infected with wild type vaccinia virusformed VLPs containing L1 protein (derived from VV pSX1iL1) and L1 andL2 protein (derived from VV pSX11L1/L2). In similar manner w pSX6L1 andVV pSX6L1/L2 after transfection of a host cell produced VLPs containingHPV6b L1 and VLPs containing HPV6b L1 and HPV6b L2.

It also will be appreciated that the invention includes within its scopeviruses doubly recombinant for papillomaviruses capsid proteins L1 andL2 as well as recombinant viruses containing papillomavirus capsidprotein L1.

It will further be appreciated that the invention includes within itsscope a method of diagnosis of papilloma virus infection by ELISAincluding the step of detection of VLP particles containing proteins L1and L2.

TABLE 1 15 AA overlapping peptides from the predicted sequence of theHPV16 L1 protein SEQ ID NO. SEQUENCE RANGE 1 MQVTFIYILVITCYE  (1-15) 2ITCYENDVNVYHIFF (11-25) 3 YHIFFQMSLWLPSEA (21-35) 4 LPSEATVYLPPVPVS(31-45) 5 PVPVSKVVSTDEYVA (41-55) 6 DEYVARTNIYYHAGT (51-65) 7YHAGTSRLLAVGHPY (61-75) 8 VGHPYFPIKKPNNNK (71-85) 9 PNNNKILVPKVSGLQ(81-95) 10 VSGLQYRVFRIHLPD  (91-105) 11 IHLPDPNKFGFPDTS (101-115) 12FPDTSFYNPDTQRLV (111-125) 13 TWRLVWACVGVEVGR (121-135) 14VEVGRGQPLGVGISG (131-145) 15 VGISGHPLLNKLDDT (141-155) 16KLDDTENASAYAANA (151-165) 17 YAANAGVDNRECISM (161-175) 18ECISMDYKQTQLCLI (171-185) 19 QLCLIGCKPPIGEHW (191-195) 20IGEHWGKGSPCTNVA (191-205) 21 CTNVAVNPGDCPPLE (101-215) 22CPPLELINTVIQDGD (211-225) 23 IQDGDMVHTGFGAMD (221-235) 24FGAMDFTTLQANKSE (231-245) 25 ANKSEVPLDICTSIC (241-255) 26CTSICKYPDYIKMVS (251-265) 27 IKMVSEPYGDSLFFY (261-275) 28SLFFYLRREQMFVRH (271-285) 29 MFVRHLFNRAGTVGE (281-295) 30GTVGENVPDDLYIKG (291-305) 31 LYIKGSGSTANLASS (301-315) 32NLASSNYFPTPSGSM (311-325) 33 PSGSMVTSDAQIFNK (321-335) 34QIFNKPYWLQRAQGH (331-345) 35 RAQGHNNGICWGNQL (341-355) 36WGNQLFVTVVDTTRS (351-365) 37 DTTRSTNMSLCAAIS (361-375) 38CAAISTSETTYKNTN (371-385) 39 YKNTNFKEYLRHGEE (381-395) 40RHGEEYDLQFIFQLC (391-405) 41 IFQLCKITLTADVMT (401-415) 42ADVMTYIHSMNSTIL (411-425) 43 NSTILEDWNFGLQPP (421-435) 44GLQPPPGGTLEDTYR (431-445) 45 EDTYRFVTQAIACQK (441-455) 46IACQKHTPPAPKEDD (451-465) 47 PKEDDPLKKYTFWEV (461-475) 48 TFWEVNLKEKFSADL (471-485) 49 FSADLDQFPLGRKFL (481-495) 50 GRKFLLQAGLKAKPK(491-505) 51 KAKPKFTLGKRKATP (501-515) 52 RKATPTTSSTSTTAK (511-525) 53STTAKRKKRKL (521-531)The sequence of each L1 peptide is give using the single letter code.The location of each peptide within the HPV16 L1 protein is given,assigning position 1 to the N terminal methionine. The short C terminalpeptide (no 53) was not used for these experiments.

TABLE 2 Immunoreactivity to virus-like particles of sera from miceimmunized with synthetic HPV16 capsids. Reactivity with virus-likeparticles Mice immunized with VLPs Immunised with Mouse strain Expt 1 (n= 5) Expt 2 (n = 5) nn2 CFA BALB/C  1.06 ± 1.29* 1.71 ± 0.06 0.36 ± 0.04B10A 1.08 ± 0.06 1.75 ± 0.04 0.24 ± 0.02 C57B1/6 0.86 ± 0.20 1.70 ± 0.120.25 ± 0.03 *Values are OD 415 units, and are given as the mean ± 1standard deviation

1. An isolated HPV-16 L1 protein encoded by a polynucleotide in whichthe first methionine codon of said polynucleotide corresponds to thesecond ATG codon of an HPV-16 L1 gene, wherein said HPV-16 L1 protein iscapable of forming a virus-like particle when expressed with HPV-16 L2protein.
 2. An immunogenic composition that comprises the HPV-16 L1protein of claim
 1. 3. The immunogenic composition of claim 2, furthercomprising an adjuvant.
 4. A virus-like particle that comprises theHPV-16 L1 protein of claim
 1. 5. An immunogenic composition thatcomprises the virus-like particle of claim
 4. 6. The immunogeniccomposition of claim 5, further comprising an adjuvant.
 7. An isolatedHPV-16 L1 protein encoded by a 1518 base pair polynucleotide in whichthe first methionine codon of said polynucleotide corresponds to thesecond ATG codon of an HPV-16 L1 gene, wherein said HPV-16 L1 protein iscapable of forming a virus-like particle when expressed with HPV-16 L2protein.
 8. An immunogenic composition that comprises the HPV-16 L1protein of claim
 7. 9. The immunogenic composition of claim 8, furthercomprising an adjuvant.
 10. A virus-like particle that comprises theHPV-16 L1 protein of claim
 7. 11. An immunogenic composition thatcomprises the virus-like particle of claim
 10. 12. The immunogeniccomposition of claim 11, further comprising an adjuvant.
 13. An isolatedHPV-16 L1 protein encoded by a polynucleotide consisting essentially ofa polynucleotide segment that begins at nucleotide 5637 of the HPV16genome, wherein said HPV-16 L1 protein lacks the N-terminal amino acidsequence encoded by nucleotides 5559 to 5636 of the HPV-16 genome and iscapable of forming a virus-like particle when expressed with HPV-16 L2protein.
 14. An immunogenic composition that comprises the HPV-16 L1protein of claim
 13. 15. The immunogenic composition of claim 14,further comprising an adjuvant.
 16. A virus-like particle that comprisesthe HPV-16 L1 protein of claim
 13. 17. An immunogenic composition thatcomprises the virus-like particle of claim
 16. 18. The immunogeniccomposition of claim 17, further comprising an adjuvant.
 19. An isolatedHPV-16 L1 protein that has the N-terminal amino acid sequence MSLWLPSEA(amino acid residues 7-15 of SEQ ID NO: 3) or SLWLPSEA (amino acidresidues 8-15 of SEQ ID NO: 3), wherein said HPV-16 L1 protein iscapable of forming a virus-like particle when expressed with HPV-16 L2protein.
 20. An immunogenic composition that comprises the HPV-16 L1protein of claim
 19. 21. The immunogenic composition of claim 20,further comprising an adjuvant.
 22. A virus-like particle that comprisesthe HPV-16 L1 protein of claim
 19. 23. An immunogenic composition thatcomprises the virus-like particle of claim
 22. 24. The immunogeniccomposition of claim 23, further comprising an adjuvant.
 25. An isolatedHPV-16 L1 protein that lacks the N-terminal amino acid sequence encodedby nucleotides 5559 to 5636 of the HPV-16 genome, wherein said HPV-16 L1protein is capable of forming a virus-like particle when expressed withHPV-16 L2 protein.
 26. An immunogenic composition that comprises theHPV-16 L1 protein of claim
 25. 27. The immunogenic composition of claim26, further comprising an adjuvant.
 28. A virus-like particle thatcomprises the HPV-16 L1 protein of claim
 25. 29. An immunogeniccomposition that comprises the virus-like particle of claim
 28. 30. Theimmunogenic composition of claim 29, further comprising an adjuvant.